The study on microorganism sensitivity to antimicrobial agents is one of the most important functions of clinical microbiology laboratories. The study is conducted by means of sensitivity testings or antibiogram, the main objective of which is to evaluate in the laboratory the response of a microorganism to one or several antimicrobial agents.
Some of the most commonly used methods in daily clinical practice include (i) diffusion methods such as disc-dish antibiogram based on the work of Bauer, Kirby et al. (Bauer A W, et al. Am. J. Clin. Pathol. 1966, 45:493-496) or the Epsilon-test or E-test method (AB Biodisk, Sweden), or (ii) dilution methods such as agar dilution method or broth microdilution method. By comparing diffusion methods to dilution methods, the latter are more technically complex and almost always more expensive, particularly when commercial microdilution panels are used. Microdilution methods in liquid medium are the most commonly used methods in routine clinical microbiology laboratory practice.
In many laboratories, the use of commercial panels is based on the use of semi-automatic incubation-reading-interpretation systems; this facilitates their use but has the drawback of an increased expenditure. Some companies have introduced on the market panels wherein the culture medium includes a fluorescent indicator that allows quickly obtaining (less than 8 hours) the results. In relation to the rapid determination of resistance to antibiotics acting on the bacterial wall, such as β-lactams, a fluorogenic compound that can be metabolized is added to the culture medium (patent application WO/1992/019763). If the organism grows with the antibiotic, the metabolism of the bacterium leads to the release of the fluorophore. If the organism does not grow, the fluorescence of the sample increases.
Another possibility is to use a color indicator compound, such as tetrazolium, which causes a color change after adding an electron carrier, such as phenazine methosulfate, in the event of sensitivity to the antibiotic. However, there are still not enough data to allow suggesting the regular use of such panels.
Several commercial companies are also evaluating expert systems (software) which facilitate the clinical interpretation of the obtained results; it is safe to assume that such systems will be widely used in the future. Several systems are available on the market today, MicroScan WalkAway, Vitek and Wider being the most outstanding ones. In the absence of greater clinical experience with the use of fluorescent indicators, the mean response time for obtaining the susceptibility of a specific microorganism to antimicrobial agents ranges, such as in the diffusion methods mentioned above, between 18 and 24 hours. Recently, the possibility of using a dielectrophoresis system that detects changes in the electrophysiology of the cell after administering the antibiotic has been pointed out (Hoettges K F, Dale J W, Hughes M P. Rapid determination of antibiotic resistance in E. coli using dielectrophoresis. Phys Med Biol 2007; 52:6001-6009).
Another development for antibiotics acting on the bacterial wall, such as β-lactams, consists of detecting by means of a specific substrate the activity of cytoplasmic enzymes that are released by the cell to the medium, if the antibiotic has been effective (European Patent EP0135023).
BACcelr8r™ is a platform under development by Accelr8 for automatically identifying microorganisms and studying their resistance to antibiotics. It does not use culture, the isolation of bacteria is not necessary either. It works by means of cassettes wherein each cassette corresponds to a sample. It uses an automated system with a microscope controlled by means of a computer, a digital camera and an analysis software. A pump maintains a flow of bacteria-containing medium in different conditions through the cassette. The antibiotic resistance analysis could be completed in 8 hours.
Patent application US2004/0014066 describes a method for detecting in a sample the activity of an antibiotic affecting cell integrity which comprises (a) providing a transformed microorganism comprising a nucleic acid encoding a promoter which is operably linked to a heterologous reporter gene capable of emitting a detectable signal, and (b) contacting the sample with the transformed microorganism, (c) observing said microorganism for said detectable signal, wherein the promoter is regulated by a two-component signal transduction system, wherein the components are (i) a receptor sensitive to changes in the cell envelope or membrane of the microorganism and (ii) a trans-acting factor which is activated in response to a stimulation by the receptor and which regulates the promoter.
Antibiotics Acting on the Bacterial Wall
The skeleton of bacterial cell wall is made up of a heteropolymer, the murein peptidoglycan. This macromolecule is formed by an alternating sequence of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) bound to one another by means of β-1,4 bonds. The chain is a straight, unbranched chain forming the basic structure of the cell wall. The N-acetylmuramic acid has a lactic acid group linking with a short peptide chain (tetrapeptide). The amino acids typical of this chain include L-alanine, D-glutamic acid, m-diaminopimelic acid or L-lysine or D-alanine.
The antibiotics which inhibit bacterial wall synthesis are different families of drugs acting on the different steps of bacterial wall synthesis:                cycloserine is a D-alanine analog and competitively inhibits the binding of this amino acid to the enzymes D-alanine-D-alanine synthetase and alanine racemase, preventing them from binding to the precursors of peptidoglycan.        fosfomycin blocks the synthesis of the precursors of peptidoglycan.        bacitracin inhibits the recycling of undecaprenyl, the lipid carrier which carries peptidoglycan to the outside of the cell.        the glucopeptide or glycopeptide antibiotics are a class of peptides with sugars bound thereto, such as in the bacterial cell wall, having a high affinity for the precursors of this structure. The most well known glycopeptide antibiotics are vancomycin and teicoplanin. Vancomycin performs its bactericidal action by inhibiting bacterial cell wall synthesis, binding to the D-alanine-D-alanine (D-Ala-D-Ala) fragment of the pentapeptide on the wall of Gram+ bacteria, blocking the incorporation of peptides on the cell wall. Secondarily, vancomycin would act through other mechanisms such as disrupting cytoplasmic membrane permeability and inhibiting RNA synthesis, which is performed after the drug has bound to the peptidoglycan.        the β-lactam antibiotics perform bactericidal function by interfering the transverse binding or interpeptide bridge necessary for cross-linking. They inhibit the activity of PBPs, serine proteases or transpeptidases, by binding to them in an irreversible manner.        other antibiotics interfering with wall synthesis are isoniazid, ethionamide and ethambutol. Like the cycloserine mentioned above, they are used in the treatment of mycobacterial infections. Isoniazid has bactericidal activity in the active replication phase. It affects mycolic acid synthesis, interrupting the elongation of fatty acids. Ethionamide also inhibits mycolic acid synthesis. Ethambutol interferes with cell wall arabinogalactan synthesis. The resistance to these antibiotics is due to the lack of penetration into the bacterium and/or modification of their cellular targets.        
Antibiotic resistance causes tens of thousands of deaths every year. Many of these deaths could be avoided with an antibiotic treatment properly selected for effectiveness. Given the levels of resistance, it is necessary to prepare the bacterial culture, followed by an antibiogram. To complete the foregoing, the bacteria must usually grow for 2-3 days. The antibiogram itself usually requires at least one day of incubation, for common fast-growing bacteria.
For patients in critical condition in ICU, a rapid antibiotic treatment is important. Given the delay of the antibiogram, it is performed empirically. Such treatment is ineffective in 20-40% of the cases, and the change of treatment after the results of the antibiogram may no longer be effective. In this situation, it is important to have a rapid antibiogram system. The antibiotics acting on the bacterial wall, specifically β-lactams, are a very large group and the most commonly used in anti-infective therapy. It is of great interest to have rapid antibiogram systems for such antibiotics.